Vaccine and Infectious Disease Organization (VIDO)-International Vaccine Centre (InterVac), University of Saskatchewan, Saskatoon, Saskatchewan, S7N 5E3, Canada; School of Public Health, Vaccinology and Immunotherapeutics, Saskatchewan, Canada.
Large Animal Clinical Sciences (LACS), Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, Saskatchewan, S7N 5B4, Canada.
Vet Microbiol. 2019 Aug;235:270-279. doi: 10.1016/j.vetmic.2019.07.014. Epub 2019 Jul 19.
Lawsonia intracellularis is an obligate intracellular microorganism and the causative agent of porcine proliferative enteropathy. Due to its obligate intracellular nature, characterization of antigens and proteins involved in host-pathogen interaction and immune recognition have been difficult to achieve using conventional microbiological techniques. In this work, we used 2-dimensional gel electrophoresis coupled with Western-immunoblotting, mass spectrometry and bioinformatics to identify bacterial proteins that interact in vitro with pig intestinal cells (IPEC-1), have immunogenic properties and the potential to be used as subunit vaccine antigens. We detected eleven immunogenic bacterial proteins from which fliC (LI0710), LI1153 (annotated by NCBI as Putative protein N), and LI0649 (annotated as autotransporter) were predicted to be expressed on the outer membrane while LI0169 (oppA; annotated as ABC dipeptide transport system) was predicted to be periplasmic with a transmembrane domain forming a central pore through the plasma membrane. Genes coding for these four proteins were cloned and expressed in Escherichia coli and the corresponding recombinant proteins were purified using affinity chromatography. Porcine hyperimmune serum against whole Lawsonia lysate established that all four recombinant proteins were immunogenic. Further, rabbit hyperimmune sera generated against the vaccine strain of L. intracellularis and rabbit serum specific for each recombinant protein showed an inhibitory effect on the attachment and penetration of live, avirulent L. intracellularis, thus indicating that each protein is a potential neutralizing antibody target and a candidate for subunit vaccine formulation.
内罗毕细胞质体是一种严格的细胞内微生物，也是猪增生性肠炎的病原体。由于其严格的细胞内特性，使用传统的微生物学技术来描述参与宿主-病原体相互作用和免疫识别的抗原和蛋白质一直具有挑战性。在这项工作中，我们使用二维凝胶电泳结合 Western 免疫印迹、质谱和生物信息学来鉴定体外与猪肠细胞（IPEC-1）相互作用、具有免疫原性且有可能用作亚单位疫苗抗原的细菌蛋白。我们从 11 种免疫原性细菌蛋白中检测到 fliC（LI0710）、LI1153（由 NCBI 注释为推定蛋白 N）和 LI0649（注释为自转运体），它们被预测在外膜上表达，而 LI0169（oppA；注释为 ABC 二肽转运系统）被预测在周质中，具有跨膜结构域，形成穿过质膜的中央孔。这些蛋白的编码基因在大肠杆菌中进行了克隆和表达，并使用亲和层析对相应的重组蛋白进行了纯化。针对整个 Lawsonia 裂解物的猪高免血清证实了这四种重组蛋白均具有免疫原性。此外，针对 L. intracellularis 疫苗株产生的兔高免血清和针对每种重组蛋白的兔血清对活的、无毒力的 L. intracellularis 的附着和穿透具有抑制作用，这表明每种蛋白都是潜在的中和抗体靶标，也是亚单位疫苗制剂的候选物。
